Project Summary/Abstract The function and fate of any given cell is determined by the gene expression profile of that cell. While transcription plays a key role in determining gene expression, post-transcriptional regulatory events also are of vital importance in determining the spatial and temporal pattern of gene expression. One crucial post- transcriptional step is addition of a polyadenosine or poly(A) tail following 3'-end cleavage of mRNA. This poly(A) tail modulates numerous events including mRNA export from the nucleus, translation, transport and turnover, largely via the recruitment of polyadenosine RNA binding proteins (Pabs). Polyadenylation of additional classes of RNA has also been implicated in quality control. The critical importance of proper polyadenylation and Pab protein function is evident in the number of human diseases that arise due to mutations in genes encoding these proteins. In the previous funding cycle, we collaborated to identify mutations in the human ZC3H14 gene, which encodes a ubiquitously expressed, evolutionarily conserved, zinc finger, nuclear polyadenosine RNA binding protein (Pab), that cause an autosomal recessive form of non- syndromic intellectual disability (previously termed mental retardation). These patients have severely impaired brain function with IQs in the range of 35-50 as compared to an average adult IQ range of 90-110. Although little is known about the function of ZC3H14, studies of the budding yeast (Nab2) and Drosophila counterparts (dNab2) provide compelling evidence that this class of proteins plays an evolutionarily conserved role in regulating poly(A) tail length. The broad, long-term objective of this proposal is to define how poly(A) tail length is regulated by the zinc finger Pabs to understand both the critical role in post-transcriptional regulation of gene expression and the mechanisms underlying neuronal defects in affected patients. This proposal exploits exciting preliminary data collected through studies of this evolutionarily conserved class of proteins in budding yeast, Drosophila, and cultured neuronal cells capitalizing on established tools and models. These studies strongly implicate the complex of 3'-5 riboexonucleases known as the nuclear exosome in cooperating with Nab2 to regulate poly(A) tail length. Based on preliminary data, we will test the hypothesis that the Nab2 class of RNA binding proteins cooperates with the exosome to regulate poly(A) tail length of RNA thus ensuring proper neuronal function. This hypothesis will be tested through three complementary Specific Aims that seek to: 1) define a molecular mechanism for Nab2/ZC3H14 in regulating poly(A) tail length (Aim 1); extend these studies into our established Drosophila model to link molecular mechanisms to neuronal function (Aim 2); and finally determine whether specific RNAs or classes of RNAs accumulate extended poly(A) tails upon loss of Nab2/ZC3H14 function (Aim 3). Successful completion of these aims will provide insight into a critical point in post-transcriptional regulation of gene expression mediated by a recently described family of Pab proteins and also lend insight into the molecular defects underlying intellectual disability in patients.